Preface
The origins of liposome research can be traced to the contributions by Alec
Bangham and colleagues in the mid 1960s. The description of lecithin disper-
sions as containing ‘‘spherulites composed of concentric lamellae’’ (A.D.
Bangham and R.W. Horne, J. Mol. Biol. 8, 660, 1964) was followed by the
observation that ‘‘the diffusion of univalent cations and anions out of spontan-
eously formed liquid crystals of lecithin is remarkably similar to the diffusion of
such ions across biological membranes (A.D. Bangham, M.M. Standish and
J.C. Watkins, J. Mol. Biol. 13, 238, 1965). Following early studies on the
biophysical characterization of multilamellar and unilamellar liposomes, inves-
tigators began to utilize liposomes as a well-defined model to understand the
structure and function of biological membranes. It was also recognized by
pioneers including Gregory Gregoriadis and Demetrios Papahadjopoulos that
liposomes could be used as drug delivery vehicles. It is gratifying that their
efforts and the work of those inspired by them have lead to the development of
liposomal formulations of doxorubicin, daunorubicin and amphotericin B now
utilized in the clinic. Other medical applications of liposomes include their use
as vaccine adjuvants and gene delivery vehicles, which are being explored in
the laboratory as well as in clinical trials. The field has progressed enormously
in the 38 years since 1965.
This volume includes applications of liposomes in immunology, diagnostics,
and gene delivery and gene therapy. I hope that these chapters will facilitate
the work of graduate students, post-doctoral fellows, and established scientists
entering liposome research. Other volumes in this series cover additional
subdisciplines in liposomology.
The areas represented in this volume are by no means exhaustive. I have
tried to identify the experts in each area of liposome research, particularly
those who have contributed to the field over some time. It is unfortunate that I
was unable to convince some prominent investigators to contribute to the
volume. Some invited contributors were not able to prepare their chapters,
despite generous extensions of time. In some cases I may have inadvertently

overlooked some experts in a particular area, and to these individuals I extend
my apologies. Their primary contributions to the field will, nevertheless, not go
unnoticed, in the citations in these volumes and in the hearts and minds of the
many investigators in liposome research.
xv
I would like to express my gratitude to all the colleagues who graciously
contributed to these volumes. I would like to thank Shirley Light of Academic
Press for her encouragement for this project, and Noelle Gracy of Elsevier
Science for her help at the later stages of the project. I am especially thankful to
my wife Diana Flasher for her understanding, support and love during the
seemingly never-ending editing process, and my children Avery and Maxine
for their unique curiosity, creativity, cheer, and love. Finally, I wish to dedicate
this volume to two other members of my family who have been influential in
my life, with their love and support since my childhood days, my aunt Sevim
Uygurer and my brother Dr. Arda Du
¨
zgu
¨
nes,
Nejat Du
¨
zgu
¨
nes,
xvi preface
METHODS IN ENZYMOLOGY
EDITORS-IN-CHIEF
John N. Abelson Melvin I. Simon
DIVISION OF BIOLOGY
CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA
FOUNDING EDITORS
Sidney P. Colowick and Nathan O. Kaplan
Contributors to Volume 373
Article numbers are in parentheses and following the names of contributors.
Affiliations listed are current.
Salvador F. Alin
˜
o (26), Departamento de
Famacologia, Facultad de Medicina, Uni-
versidad de Valencia, Avda Blasco Ibanez
15, 46010 Valencia, Spain
Carl R. Alving (2, 3, 10), Department of
Membrane Biochemistry, Walter Reed
Army Institute of Research, Washington,
D.C. 20307
M. A. Arangoa (22), Department of
Pharmacology and Pharmaceutical
Technology, School of Pharmacy, Univer-
sity of Navarra, 31080 Pamplona, Spain
Udo Bakowsky (18), Department of
Pharmaceutical Technology and Biophar-
macy, University of Saarbruecken,
Germany
Richard R. Bankert (33), Department of
Microbiology, SUNY at Buffalo, 138
Farber Hall, 3435 Main Street, Buffalo,
New York 14214
Lajos Baranyi (10), Department of Mem-
brane Biochemistry, Walter Reed Army

Institute of Research, Washington, D.C.
20307
Marta Benet (26), Departamento de Fa-
macologia, Facultad de Medicina, Univer-
sidad de Valencia, Avda Blasco Ibanez 15,
46010 Valencia, Spain
Michael Bodo (10), Department of Mem-
brane Biochemistry, Walter Reed Army
Institute of Research, Washington, D.C.
20307
Otto C. Boerman (15), Department of Nu-
clear Medicine (565), University Medical
Center Nijmegen, P.O. Box 9101, 6500
HB Nijmegen, The Netherlands
Elena Bogdanenko (28), V N Orekhovich
Institute of Biomedical Chemistry, Rus-
sian Academy of Medical Sciences, 10,
Pogodinska ya Street, 119832 Moscow,
Russia
Jeff W.M. Bulte (12), Department of
Radiology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland
21205
Laura Bungener (5), Department of Med-
ical Microbiology, Molecular Virology
Section, University of Groningen, 9713
AV Groningen, The Netherlands
Rolf Bunger (10), Department of
Membrane Biochemistry, Walter Reed
Army Institute of Research, Washington,

D.C. 20307
Gerardo Byk (23), Laboratory of Peptido-
mimetics and Genetic Chemistry, Bar Ilan
University, Department of Chemistry,
52900 Ramat Gan, Israel
Jin-Soo Chang (9), Morgan Biotechnology
Research Institute, 341 Pojung-Ri, Koon-
sung-Myon, Youngin City, Kyonggi-Do
449-910, South Korea
Myeong-Jun Choi (9), Charmzone
Co.,Ltd., Bioimaterial Research Center,
301 Hankang Building, 184-11 Kwang-
jang-dong, Kwangjin-ju, Seoul, Korea
Jaime Crespo (26), Departamento de Fa-
macologia, Facultad de Medicina, Univer-
sidad de Valencia, Avda Blasco Ibanez 15,
46010 Valencia, Spain
Toos Daemen (5), Department of Medical
Microbiology, Molecular Virology
Section, University of Groningen, 9713
AV Groningen, The Netherlands
ix
Sumeet Dagar (13), Departments of
Pharmaceutics and Pharmacodynamics,
University of Illinois at Chicago, 833
Wood Street, Chicago, Illinois 60612
Francisco Dası
´
(26), Departamento de Fa-
macologia, Facultad de Medicina, Univer-

sidad de Valencia, Avda Blasco Ibanez 15,
46010 Valencia, Spain
Robert J. Debs (34), Geraldine Brush
Cancer Research Institute, 2330 Clay
Street, San Francisco, California 94115
Marcel De Cuyper (12), Interdisciplinary
Research Center, Katholieke Universiteit
Leuven, Campus Kortrijk, B-8500
Kortrijk, Belgium
C. Tros De Ilarduya (22), Department of
Pharmacology and Pharmaceutical Tech-
nology, School of Pharmacy, University
of Navarra, 31080 Pamplona, Spain
Nejat Du
¨
zgu
¨
nes, (19, 22, 24, 28), Depart-
ment of Microbiology, University of the
Pacific School of Dentistry, 2155 Webster
Street, San Francisco, California 94115
Nejat K. Eg
ˇ
ilmez (33), Department of
Microbiology, SUNY at Buffalo, 138
Farber Hall, 3435 Main Street, Buffalo,
New York 14214
Abdelatif Elouahabi (20), Center for
Structural Biology and Bioinformatics,
CP 206/2, Campus Plaine-ULB, Blv du

Triomphe, 1050 Brussels, Belgium
Henrique Faneca (19), Department of
Biochemistry, Faculty of Sciences and
Technology, University of Coimbra, 3000
Coimbra, Portugal
Sylvia Fong (34), Geraldine Brush Cancer
Research Institute, 2330 Clay Street, San
Francisco, California 94115
Benoı
ˆ
t Frisch (4), Laboratoire de Chimie
Bioorganique, UMR 7514 CNRS-ULP,
Faculte de Pharmacie, 74 Route du Rhin,
Illkirch 67400, France
Stephen J. Frost (16), Department of Clin-
ical Biochemistry, The Princess Royal
Hospital, Lewes Rd. Haywards Heath,
West Suxxes RH16 3LU, England
M. Teresa Gira
˜
o Da Cruz (24), Depart-
ment of Biochemistry, Faculty of Sciences
and Technology, University of Coimbra,
3000 Coimbra, Portugal
Laurent Giraudo (7), Centre d’Immuno-
logie de Marseille-Luminy, Campus de
Luminy, Case 906, 13288 Marsielle
Cedex 09, France
Mitsuru Hashida (25), Graduate School
of Pharmaceutical Sciences, Kyoto Uni-

versity, Sakyo-ku, Kyoto 606-850, Japan
Kazuya Hiraoka (30), Division of Gene
Therapy Science, Graduate School of
Medicine, Osaka University, 2-2 Yama-
da-oka, Suita City, Osaka 565-0871,
Japan
Dick Hoekstra (18), Department of Mem-
brane Cell Biology, University of Gron-
ingen, Antonius Deusinglaan 1, 9713 AV
Groningen, The Netherlands
Leaf Huang (21), Center for Pharmacoge-
netics, School of Pharmacy, University of
Pittsburgh, 633 Salk Hall, Pittsburgh,
Pennsylvania 15213
Anke Huckreide (5), Department of Med-
ical Microbiology, Molecular Virology
Section, University of Groningen, 9713
AV Groningen, The Netherlands
Yasufumi Kaneda (30), Division of Gene
Therapy Science, Graduate School of
Medicine, Osaka University, 2-2 Yamada-
oka, Suita City, Osaka 565-0871, Japan
Shigeru Kawakami (25), Faculty of
Pharmaceutical Sciences, Nagasaki
University, Magaski 852-8521, Japan
Chong-Kook Kim (17), College of Phar-
macy, Seoul National University, San
56-1, Shinlim-Doug, Kwanak-Gu, Seoul,
South Korea
x contributors to volume 373

Kenji Kono (27), Department of Applied
Materials Science, Graduate School of
Engineering, Osaka Prefecture Univer-
sity, 1-1, Gakuencho, Sakai, Osaka
599-8531, Japan
Krystyna Konopka (31), Department of
Microbiology, University of the Pacific
School of Dentistry, 2155 Webster Street,
San Francisco, California 94115
Lakshmi Krishnan (11), Institute for Bio-
logical Sciences, National Research
Council of Canada, 100 Sussex Drive,
Ottawa, Ontario K1A 0R6, Canada
Peter E. Jensen (8), Department of Path-
ology and Laboratory Medicine, Emory
University School of Medicine, Atlanta,
Georgia 30322
Lawrence B. Lachman (6), Department of
Bioimmunotherapy, Box 422, The Univer-
sity of Texas MD Anderson Cancer
Center, 1515 Holcombe Blvd., Houston,
Texas, 77030
Olivier Lambert (29), Institut Curie,
Section de Recherche, UMR-CNRS 168
et LRC-CEA 8, 11 rue Pierre et Marie
Curie, 75231 Paris, France
Peter Laverman (15), Department of Nu-
clear Medicine (565), University Medical
Center Nijmegen, P.O. Box 9101, 6500
HB Nijmegen, The Netherlands

Paul J. Lee (14), Vitreoretinal Surgical
Fellow, Tulane University Medical
Center, 1430 Tulane Avenue, New
Orleans, Louisiana 70112
Lee Leserman (7), Centre d’Immunologie
de Marseille-Luminy, Campus de Lu-
miny, Case 906, 13288 Marsielle Cedex
09, France
Song Li (21), Center for Pharmacogenetics,
School of Pharmacy, University of Pitts-
burgh, 633 Salk Hall, Pittsburgh, Penn-
sylvania 15213
Soo-Jeong Lim (17), College of Pharmacy,
Seoul National University, San 56-1,
Shinlim-Doug, Kwanak-Gu, Seoul, South
Korea
Yong Liu (34), Geraldine Brush Cancer
Research Institute, 2330 Clay Street,
San Francisco, California 94115
Patrick Machy (7), Centre d’Immunologie
de Marseille-Luminy, Campus de Lu-
miny, Case 906, 13288 Marsielle Cedex
09, France
Miguel Mano (19), Department of Bio-
chemistry, Faculty of Sciences and Tech-
nology, University of Coimbra, 3000
Coimbra, Portugal
Gary R. Matyas (3), Department of Mem-
brane Biochemistry, Walter Reed Army
Institute of Research, Washington, D.C.

20307
Nathalie Mignet (23), UMR 7001, Labor-
atoire de Chimie Bioorganiquet et de Bio-
technologie Moleculaire et Cellulaire,
Ecole National Superieure de Chimie de
Paris, 13 Quai Jules Guesde, BP 14,
94403 Vitry sur Siene, France
Janos Milosevits (10), Department of
Membrane Biochemistry, Walter Reed
Army Institute of Research, Washington,
D.C. 20307
Alexey Moskovtsev (28), V N Orekhovich
Institute of Biomedical Chemistry, Rus-
sian Academy of Medical Sciences, 10,
Pogodinska ya Street, 119832 Moscow,
Russia
Jean M. Muderhwa (3), Department of
Membrane Biochemistry, Walter Reed
Army Institute of Research, Washington,
D.C. 20307
Makiya Nshikawa (25), Graduate School
of Pharmaceutical Sciences, Kyoto Uni-
versity, Sakyo-ku, Kyoto 606-850, Japan
contributors to volume 373 xi
Volker Oberle (18), Department of Mem-
brane Cell Biology, University of Gron-
ingen, Antonius Deusinglaan 1, 9713 AV
Groningen, The Netherlands
Hayat O
¨

nkyu
¨
ksel (13), Departments of
Pharmaceutics and Pharmacodynamics,
University of Illinois at Chicago, 833
Wood Street, Chicago, Illinois 60612
Bu
¨
lent O
¨
zpolat (6), Department of Bioim-
munotherapy, Box 422, The University of
Texas MD Anderson Cancer Center, 1515
Holcombe Blvd., Houston, Texas, 77030
William M. Pardridge (32), University of
California-Los Angeles, Warren Hall, 13-
164, 900 Veteran Avenue, Los Angeles,
California 90024
Girishchandra B. Patel (11), Institute for
Biological Sciences, National Research
Council of Canada, 100 Sussex Drive,
Ottawa, Ontario K1A 0R6, Canada
Ve
´
ronique Pector (20), Center for Struc-
tural Biology and Bioinformatics, CP
206/2, Campus Plaine-ULB, Blv du
Triomphe, 1050 Brussels, Belgium
Maria C. Pedroso De Lima (19, 24), De-
partment of Biochemistry, Faculty of Sci-

ences and Technology, University of
Coimbra, 3000 Coimbra, Portugal
Nuno Penacho (19), Department of Bio-
chemistry, Faculty of Sciences and Tech-
nology, University of Coimbra, 300
Coimbra, Portugal
Gholam A. Peyman (14), Ophthalmology
Department (SL 69), Tulane University
Medical Center, 1430 Tulane Avenue,
New Orleans, Louisiana 70112
Pedro Pires (24), Center for Neuroscience
and Cell Biology, University of Coimbra,
3000 Coimbra, Portugal
Olga Podobed (28), V N Orekhovich Insti-
tute of Biomedical Chemistry, Russian
Academy of Medical Sciences, 10, Pogo-
dinska ya Street, 119832 Moscow, Russia
Mangala Rao (2), Department of Mem-
brane Biochemistry, Walter Reed Army
Institute of Research, Washington, D.C.
20307
Jean-Louis Rigaud (29), Institut Curie,
Section de Recherche, UMR-CNRS 168
et LRC-CEA 8, 11 rue Pierre et Marie
Curie, 75231 Paris, France
Audrey Roth (4), Laboratoire de Chimie
Bioorganique, UMR 7514 CNRS-ULP,
Faculte de Pharmacie, 74 Route du Rhin,
Illkirch 67400, France
Stephen W. Rothwell (2), Department of

Membrane Biochemistry, Walter Reed
Army Institute of Research, Washington,
D.C. 20307
Israel Rubinstein (13), Departments of
Pharmaceutics and Pharmacodynamics,
University of Illinois at Chicago, 833
Wood Street, Chicago, Illinois 60612
Jean-Marie Ruysschaert (20), Center for
Structural Biology and Bioinformatics,
CP 206/2, Campus Plaine-ULB, Blv du
Triomphe, 1050 Brussels, Belgium
Sandor Savay (10), Department of Mem-
brane Biochemistry, Walter Reed Army
Institute of Research, Washington, D.C.
20307
Daniel Scherman (23), UMR 7001, La-
boratoire de Chimie Bioorganiquet et de
Biotechnologie Moleculaire et Cellulaire,
Ecole National Superieure de Chimie de
Paris, 13 Quai Jules Guesde, BP 14,
94403 Vitry sur Siene, France
Francis Schuber (4), Laboratoire de Chi-
mie Bioorganique, UMR 7514 CNRS-
ULP, Faculte de Pharmacie, 74 Route
du Rhin, Illkirch 67400, France
Karine Serre (7), Centre d’Immunologie de
Marseille-Luminy, Campus de Luminy,
Case 906, 13288 Marsielle Cedex 09,
France
xii contributors to volume 373

Se
´
rgio Simo
˜
es (19, 24), Department of Bio-
chemistry, Faculty of Sciences and Tech-
nology, University of Coimbra, 3000
Coimbra, Portugal
G. Dennis Sprott (11), Institute for Bio-
logical Sciences, National Research
Council of Canada, 100 Sussex Drive,
Ottawa, Ontario K1A 0R6, Canada
Gert Storm (15), Department of Pharma-
ceutics, Utrecht Institute for Pharmaceut-
ical Sciences (UIPS), Utrecht University,
The Netherlands
Janos Szebeni (10), Department of Mem-
brane Biochemistry, Walter Reed Army
Institute of Research, Washington, D.C.
20307
Toru Takagishi (27), Department of Ap-
plied Materials Science, Graduate School
of Engineering, Osaka Prefecture Univer-
sity, 1-1, Gakuencho, Sakai, Osaka
599-8531, Japan
Marc Thiry (20), Laboratory of Cell and
Tissue Biology, University of Liege, Rue
de Pitteurs, Liege, Belgium
Michel Vandenbranden (20), Center for
Structural Biology and Bioinformatics,

CP 206/2, Campus Plaine-ULB, Blv du
Triomphe, 1050 Brussels, Belgium
Esther Van Kesteren-Hendrikx (1), De-
partment of Cell and Immunology,
Faculty of Medicine, Vrije Universiteit
Medical Center, Van de Boechhorststraat
7, 1081 BT Amsterdam, The Netherlands
Nico Van Rooijen (1), Department of Cell
and Immunology, Faculty of Medicine,
Vrije Universiteit Medical Center, Van
de Boechhorststraat 7, 1081 BT Amster-
dam, The Netherlands
Larry E. Westerman (8), VGS/DVRD/
NCID, Centers for Disease Control and
Prevention, Atlanta, Georgia 30322
Barbara Wetzer (23), UMR 7001,
Laboratoire de Chimie Bioorganiquet
et de Biotechnologie Moleculaire et
Cellulaire, Ecole National Superieure de
Chimie de Paris, 13 Quai Jules
Guesde, BP 14, 94403 Vitry sur Siene,
France
Jan Wilschut (5), Department of Medical
Microbiology, Molecular Virology
Section, University of Groningen, 9713
AV Groningen, The Netherlands
Seiji Yamamoto (30), Division of Gene
Therapy Science, Graduate School of
Medicine, Osaka University, 2-2 Yama-
da-oka, Suita City, Osaka 565-0871,

Japan
Fumiyoshi Yamashita (25), Graduate
School of Pharmaceutical Sciences,
Kyoto University, Sakyo-ku, Kyoto
606-850, Japan
Jing-Shi Zhang (21), Center for Pharma-
cogenetics, School of Pharmacy, Univer-
sity of Pittsburgh, 633 Salk Hall,
Pittsburgh, Pennsylvania 15213
Renat Zhdanov (28), V N Orekhovich In-
stitute of Biomedical Chemistry, Russian
Academy of Medical Sciences, 10, Pogo-
dinska ya Street, 119832 Moscow, Russia
contributors to volume 373 xiii
[1] ‘‘In Vivo’’ Depletion of Macrophages
by Liposome-Mediated ‘‘Suicide’’
By Nico van Rooijen and Esther van Kesteren-Hendrikx
Introduction
Macrophages are multifunctional cells. They play a key role in natural
and acquired host defense reactions, in homeostasis, and in the regulation
of numerous biological processes. The main tools they use to achieve these
goals are phagocytosis followed by intracellular digestion, and production
and release of soluble mediators such as cytokines, chemokines, and nitric
oxide (NO). Macrophages can be found as resident cells in all organs of the
body, and they can be recruited to sites of inflammation. Their immediate
precursors are monocytes, which are released in the blood circulation from
the bone marrow. After some time, monocytes leave the circulation, cross
the barrier formed by the walls of blood vessels, and enter into one of the
organs, where their final differentiation into mature macrophages will take
place.

Depletion of macrophages followed by functional studies in such
macrophage-depleted animals forms a generally accepted approach to es-
tablish their role in any particular biomedical phenomenon. Early methods
for depletion of macrophages were based on the administration of silica,
carrageenan, or by various other treatments. However, incompleteness of
depletion, and even stimulation of macrophages, as well as unwanted
effects on nonphagocytic cells, were obvious disadvantages.
1
For that reason, we have developed a more sophisticated approach,
based on the liposome-mediated intracellular delivery of the bisphospho-
nate clodronate.
2,3
In this approach, liposomes are used as a Trojan horse
to get the small clodronate molecules into the macrophage. Once ingested
by macrophages, the phospholipid bilayers of liposomes are disrupted under
the influence of lysosomal phospholipases. The strongly hydrophilic clodro-
nate molecules intracellularly released in this way do not escape from
the cell, because they will not easily cross its cell membranes. As a result,
the intracellular clodronate concentration increases as more liposomes
are ingested and digested. At a certain clodronate concentration, irrevers-
ible damage causes the macrophage to be killed by apoptosis.
4,5
Clodronate
1
N. van Rooijen and A. Sanders, J. Leuk. Biol. 62, 702 (1997).
2
N. van Rooijen and R. van Nieuwmegen, Cell Tiss. Res. 238, 355 (1984).
3
N. van Rooijen and A. Sanders, J. Immunol. Meth. 174, 83 (1994).
[1] depletion of macrophages by liposomes 3

Copyright 2003, Elsevier Inc.
All rights reserved.
METHODS IN ENZYMOLOGY, VOL. 373 0076-6879/03 $35.00
molecules released in the circulation from dead macrophages will not enter
cells again, because they are not able to cross cell membranes. Moreover,
free clodronate molecules show an extremely short half-life in circulation
and body fluids. They are removed by the renal system. The combination
of low toxicity and short half-life of clodronate makes this drug the
best choice for the liposome-mediated elimination of macrophages
‘‘in vivo’’. Clodronate in its free form is used widely as a drug for the treat-
ment of malignant hypercalcemia
6
and painful bone metastasis caused by
hormone-refractory prostate cancer,
7
emphasizing its nontoxic nature.
Clodronate Liposomes in Research
Clodronate Liposomes as a Tool to Investigate Macrophage
Activities In Vivo
With the liposome-mediated macrophage ‘‘suicide’’ approach, func-
tional aspects of macrophages have been established in hundreds of studies
up to now. Many of the resulting publications are listed in the website
.
Although liposomes can not cross vascular barriers such as the walls of
capillaries, their meeting with particular macrophage populations can be
achieved by choosing the right administration routes. Among the macro-
phages that might become useful targets for manipulation by liposomes
are Kupffer cells in the liver and splenic macrophages (to be reached by
way of intravenous injection),
8

alveolar macrophages in the lungs (to be
reached by way of intratracheal instillation or intranasal administration),
9
phagocytic synovial lining cells (by means of intra-articular injection in
the synovial cavity),
10
peritoneal macrophages (by means of intraperito-
neal injection),
11
macrophages in the testis (by means of local injection),
12
4
N. van Rooijen, A. Sanders, and T. van den Berg, J. Immunol. Meth. 193, 93 (1996).
5
M. Naito, H. Nagai, S. Kawanao, H. Umezu, H. Zhu, H. Moriyama, T. Yamamoto,
H. Takatsuka, and Y. Takkei, J. Leuk. Biol. 60, 337 (1996).
6
A. List, Arch. Intern. Med. 151, 471 (1991).
7
A. Heidenreich, R. Hofmann, and U. H. Engelmann, J. Urol. 165, 136 (2001).
8
N. van Rooijen, N. Kors, M. van den Ende, and C. D. Dijkstra, Cell Tiss. Res. 260, 215
(1990).
9
T. Thepen, N. van Rooijen, and G. Kraal, J. Exp. Med. 170, 499 (1989).
10
P. L. E. M. van Lent, A. E. M. Holthuyzen, L. A. M. van den Bersselaar, N. van Rooijen,
L. A. B. Joosten, F. A. J. van de Loo, L. B. A. van de Putte, and W. B. van de Berg. Arthritis
Rheum. 39, 1545 (1996).
11

J. Biewenga, B. van de Ende, L. F. G. Krist, A. Borst, M. Chufron, and N. van Rooijen, Cell
Tiss. Res. 280, 189 (1995).
12
A. Bergh, J. E. Damber, and N. van Rooijen, J. Endocrinol. 136, 407 (1993).
4 liposomes in immunology [1]
perivascular macrophages in the central nervous system (CNS, by means of
intraventricular injection),
13
and lymph node macrophages (by means of
injection in their draining areas).
14
Depleted macrophages are replaced by new ones recruited from the
bone marrow after various periods of time. In the liver, new Kupffer cells
reappear after $5 days, and repopulation of the liver with Kupffer cells is
complete after $2 weeks. In the spleen, red pulp macrophages, marginal
metallophilic macrophages, and marginal zone macrophages reappear after
$1 week, 3 weeks, and 2 months, respectively.
15
The different repopulation
kinetics can be used to study their functional specialization. The role of
marginal zone macrophages in the antibody response to particulate anti-
gens was established in mice repopulated by red pulp macrophages
and marginal metallophilic macrophages 1 month after administration of
clodronate liposomes; in such mice, only the marginal zone macrophages
were still absent.
16
The repopulation kinetics of alveolar macrophages in
the lung, testis macrophages, lymph node macrophages, peritoneal macro-
phages, and synovium lining macrophages in joints are available in the
relevant literature.

Clodronate Liposomes in Immunodeficient Mice to Study Grafted
Human Cells In Vivo
Immunodeficient mice are widely used to harbor xenogeneic grafts of
human blood cells to study their role in host defense mechanisms, patho-
logical disorders, and diseases. However, despite the absence of functional
T and B lymphocytes as the effector cells of acquired immunity in mice
bearing for example the scid mutation, elements of the innate immune
system are still present. Macrophages are thought to form the core of
the remaining resistance against the grafted human cells.
The effects of macrophage depletion in scid mice by administration of
clodronate liposomes has been investigated in various models. Human per-
ipheral blood lymphocytes injected into macrophage-depleted scid mice
maintained a large proportion of human cells in the peripheral blood and
spleen of the mice, whereas no human cells were detected in control mice
within 72 h.
17
The minimum graft size of normal and leukemic human he-
mopoietic cells in scid mice, which results in an outgrowth of the human
13
M. M. J. Polfliet, P. H. Goede, E. M. L. van Kesteren-Hendrikx, N. van Rooijen,
C. D. Dijkstra, and T. K. van den Berg. J. Neuroimmunol. 116, 188 (2001).
14
F. G. A. Delemarre, N. Kors, G. Kraal, and N. van Rooijen, J. Leuk. Biol. 47, 251 (1990).
15
N. van Rooijen, N. Kors, and G. Kraal, J. Leuk. Biol. 45, 97 (1989).
16
F. G. A. Delemarre, N. Kors, and N. van Rooijen, Immunobiology, 182, 70 (1991).
17
C. C. Fraser, B. P. Chen, S. Webb, N. van Rooijen, and G. Kraal, Blood, 86, 183 (1995).
[1] depletion of macrophages by liposomes 5

cells in the mouse bone marrow seemed to be 10 times smaller in macro-
phage-depleted scid mice than in normal scid mice.
18
This considerable
reduction of the minimal graft size facilitates greatly studies on subsets of
human hemopoietic cells, which are not easy to obtain in large numbers.
Mice lacking the elements of acquired immunity could be made suscep-
tible to the development of the human malaria parasite Plasmodium falci-
parum by depletion of macrophages, followed by substitution of mice
erythrocytes by human red blood cells infected with P. falciparum.
19
This
new model can be used for studies on host–parasite interactions and defense
mechanisms against P. falciparum,
19
as well as in the development of anti-
malarial drugs.
20
In view of the scarcity of animals able to harbor human
parasites, this novel model offers new approaches for malaria research.
Clodronate Liposomes in Experimental Models of Therapy
Autoantibody-Mediated Disorders
Under normal circumstances, macrophages will not ingest the orga-
nism’s own particulate blood constituents. However, when autoantibodies
are produced (e.g., against platelets in immune thrombocytopenic pur-
pura [ITP] or against red blood cells [RBC] in autoimmune hemolytic
anemia [AIHA]), macrophages are responsible for the clearance of
large numbers of these autoantibody-coated platelets or RBC. As a con-
sequence, macrophages may play a key role in the induction of these
autoantibody-mediated disorders.

In a mouse model of ITP, depletion of splenic and hepatic macrophages
by liposome-encapsulated clodronate inhibited the antibody-induced
thrombocytopenia in a dose-dependent manner. Moreover, this treatment
rapidly restored the platelet counts in thrombocytopenic animals to hemato-
logical safe values, and despite additional antiplatelet antiserum treatment,
mice were able to maintain this level of platelets for at least 2 days. The
bleeding times in the treated animals were not different from those in con-
trols, demonstrating that hemostasis was well controlled in these animals.
21
18
W.Terpstra,P.J.M.Leenen,C.vandenBos,A.Prins,W.A.M.Loenen,
M. M. A. Verstegen, S. van Wijngaardt, N. van Rooijen, A. W. Wognum, G. Wagemaker,
J. J. Wielenga, and B. Lowenberg, Leukemia, 11, 1049 (1997).
19
E. Badell, C. Oeuvray, A. Moreno, S. Soe, N. van Rooijen, A. Bouzidi, and P. Druilhe,
J. Exp. Med. 192, 1653 (2000).
20
A. Moreno, E. Badell, N. van Rooijen, and P. Druilhe, Antimicrob. Agents Chemother. 45,
1847 (2001).
21
F. Alves-Rosa, C. Stranganelli, J. Cabrera, N. van Rooijen, M. S. Palermo, and M. A. Isturiz,
Blood, 96, 2834 (2000).
6 liposomes in immunology [1]
The possible application of clodronate liposomes in the treatment of
autoantibody-mediated hemolytic anemia has also been shown in a mouse
model.
22
Autoimmune hemolytic anemia (AIHA) is a disease in which
autoantibodies against RBC lead to their premature destruction. Most clin-
ically significant autoantibodies are of the IgG type, which lead primarily to

the uptake and destruction of RBC by splenic and hepatic macrophages. In
a mouse model of AIHA in which animals were given either anti-RBC
antibodies or preopsonized RBC, liposomal clodronate substantially de-
creased RBC destruction. The treatment was rapidly effective within hours
by first blocking and consecutively depleting macrophages, and its action
lasted for 1 to 2 weeks.
Rheumatoid Arthritis
Macrophages play a key role in the production of inflammatory mediators
such as cytokines, NO, and chemokines. Depletion of phagocytic lining cells
in knee joints of mice by direct injection of liposome-encapsulated clodronate
a few days before induction of arthritis with heterologous bovine type II
collagen significantly reduced the inflammatory reaction compared with con-
trols.
10
Cell influx into the synovium was decreased markedly, and expression
of interleukin-1 mRNA in the synovium was reduced strongly. Also in the
synovial washout samples, chemotactic activity was highly decreased. In add-
ition, other experiments showed that cartilage destruction was reduced in the
animals treated with clodronate liposomes.
23
Phagocytic synovial lining cells
were also involved in acute and chronic inflammation after exacerbation of
hyperreactive joints with antigen given either directly into the knee joint or
intravenously in a mouse model of antigen-induced arthritis.
24
Further human
studies revealed that a single intra-articular administration of clodronate
liposomes resulted in macrophage depletion and decreased expression of
adhesion molecules in the synovial lining of patients with long-standing
rheumatoid arthritis.

25
Transplantation
Corneal graft rejection is characterized by a massive infiltration of both
T cells and macrophages. Macrophages are found in large numbers in
rejected corneal grafts, suggesting a role for these cells in the rejection
22
M. B. Jordan, N. van Rooijen, S. Izui, J. Kappler, and P. Marrack, Blood, 101, 594 (2003).
23
P. L. E. M. van Lent, A. E. M. Holthuyzen, N. van Rooijen, L. B. A. van de Putte, and
W. B. van de Berg, Ann. Rheum. Dis. 57, 408 (1998).
24
P. L. E. M. van Lent, A. E. M. Holthuyzen, N. van Rooijen, F. A. J. van de Loo, L. B. A. van
de Putte, and W. B. van de Berg, J. Rheumatol. 25, 1135 (1998).
25
P. Barrera, A. Blom, P. L. E. M. van Lent, L. van Bloois, G. Storm, J. Beijnen, N. van
Rooijen, L. B. A. van de Putte, and W. B. van de Berg, Arthritis Rheum. 43, 1951 (2000).
[1] depletion of macrophages by liposomes 7
process. In rats treated postoperatively with subconjunctival injections of
liposome-encapsulated clodronate at the time of transplantation and sev-
eral times thereafter, grafts were not rejected during the maximum
follow-up of 100 days. Cellular infiltration in these grafts was reduced,
and there was a strong reduction in neovascularization of the cornea.
Corneal grafts in rats that had received empty liposomes were rejected
within the usual period of 17 days.
26
In additional experiments, treatment
with clodronate liposomes was shown to down-regulate local and systemic
cytotoxic T lymphocyte responses and to prevent the generation of anti-
bodies. Depletion of macrophages in the initiation phase of the immune re-
sponse seemed to induce a less vigorous attack on the grafted tissue and,

therefore, to promote graft survival.
27
Macrophage depletion by clodronate liposomes also prolonged survival
and functioning of grafts after pancreas islet xenotransplantation,
28,29
as
well as that of porcine neonatal pancreatic cell clusters contained in algin-
ate macrocapsules and transplanted into rats.
30
Treatment with clodronate
liposomes reduced markedly graft infiltration by macrophages and T cells,
and evidence has been produced that macrophages play a role in graft re-
jection by promotion of T-cell infiltration.
28
Interestingly, recent evidence
supports the idea that T-cell–activated macrophages themselves are
capable of recognizing and rejecting pancreatic islet xenografts.
31
In the
latter studies, it has been shown that CD4
þ
T cells are required for macro-
phage activation in the presence of pancreatic islet xenografts. However,
once activated, macrophages are capable of rejecting xenografts in the ab-
sence of any other effector cells; they are able to migrate to the graft site
and to identify the graft independently of other signals from T cells.
According to the authors, this suggests that in xenograft rejection, macro-
phages receive additional non-T-cell–mediated signals by way of the innate
immune system. This could explain why immunosuppressive strategies that
26

G. van der Veen, L. Broersma, C. D. Dijkstra, N. van Rooijen, G. van Rij, and R. van der
Gaag, Invest. Ophthalmol. 35, 3505 (1994).
27
T. P. A. M. Slegers, P. F. Torres, L. Broersma, N. van Rooijen, G. van Rij, and R. van der
Gaag, Invest. Ophthalmol. 41, 2239 (2000).
28
A. Fox, M. Koulmanda, T. E. Mandel, N. van Rooijen, and L. C. Harrison, Transplantation,
66, 1407 (1998).
29
G. Wu, O. Korsgren, J. Zhang, Z. Song, N. van Rooijen, and A. Tibell, Xenotransplantation,
7, 214 (2000).
30
A. Omer, M. Keegan, E. Czismadia, P. de Vos, N. van Rooijen, S. Bonner-Weir, and
G. C. Weir, Xenotransplantation 10, 240 (2003).
31
S. Yi, A. M. Lehnert, K. Davey, H. Ha, J. Kwok Wah Wong, N. van Rooijen,
W. J. Hawthorne, A. T. Patel, S. N. Walters, A. Chandra, and P. J. O’Connell,
J. Immunol. 170, 2750 (2003).
8 liposomes in immunology [1]
inhibit the alloimmune response are ineffective at suppressing T-cell–
mediated xenograft rejection.
31
Neurological Disorders
Depletion of blood-borne macrophages reduces strongly lesion forma-
tion and the development of clinical signs in experimental allergic enceph-
alomyelitis (EAE),
32
an experimental model for multiple sclerosis (MS).
Adoptive transfer of EAE with myelin basic protein–reactive CD4
þ

Tcells
to SJL/J mice was abrogated by treatment with mannosylated clodronate lipo-
somes. Invasion of the CNS by various cells was almost completely blocked
by this treatment, and the myelin sheaths appeared completely normal,
whereas marked demyelination was observed in the control groups.
33
These studies demonstrated a role for macrophages in regulating the inva-
sion of autoreactive T cells and secondary glial recruitment that ordinarily
lead to the demyelinating pathology in EAE and MS.
33
Recent studies
demonstrated that perivascular macrophages and meningeal macrophages,
constituting a major population of resident macrophages in the CNS, also
contribute to the early stages of EAE development.
13
Inflammatory mechanisms are believed to play an important role in
hyperalgesia resulting from nerve injury. It was shown that intravenous
injection of clodronate liposomes reduced the number of macrophages in
an injured nerve, alleviated thermal hyperalgesia, and protected both mye-
linated and unmyelinated fibers against degeneration. Results confirmed
the role of circulating monocytes and/or macrophages in the preservation
of myelinated axons, decreased cavitation in the development of neuro-
pathic hyperalgesia and Wallerian degeneration caused by partial nerve
injury, and it was suggested that suppression of macrophage activity
immediately after nerve injury could have some clinical potential in the
prevention of neuropathic pain.
34
Traumatic injury to the spinal cord initiates a series of destructive cellu-
lar processes that accentuate tissue damage at and beyond the original site
of trauma. The cellular inflammatory response has been implicated as one

mechanism of secondary degeneration. Within injured spinal cords of rats
treated with clodronate liposomes, macrophage infiltration was signifi-
cantly reduced at the site of impact. These animals showed marked
improvement in hindlimb use during overground locomotion. Behavioral
32
I. Huitinga, N. van Rooijen, C. J. A. de Groot, B. M. J. Uitdehaag, and C. D. Dijkstra,
J. Exp. Med. 172, 1025 (1990).
33
E. H. Tran, K. Hoekstra, N. van Rooijen, C. D. Dijkstra, and T. Owens, J. Immunol. 161,
3767 (1998).
34
T. Liu, N. van Rooijen, and D. J. Tracey, Pain, 86, 25 (2000).
[1] depletion of macrophages by liposomes 9
recovery was paralleled by a significant preservation of myelinated axons,
decreased cavitation in the rostrocaudal axis of the spinal cord, and en-
hanced sprouting and/or regeneration of axons at the site of injury. These
data implicate blood-borne macrophages as effectors of acute secondary
injury and suggest that clodronate liposomes may prove to be useful in
therapy after spinal cord injury.
35
Other Forms of T-cell–Mediated Tissue Damage
T cells seem to be responsible for liver damage in any type of acute
hepatitis. T-cell–mediated liver injury is induced for example by several
agents such as Pseudomonas exotoxin A (PEA), concanavalin A (ConA),
and by a combination of subtoxic doses of PEA and the superantigen
Staphylococcus enterotoxin B (SEB). Depletion of Kupffer cells (liver
macrophages) by clodronate liposomes protected mice from PEA-,
ConA-, or PEA/SEB–induced liver injury. In the absence of Kupffer cells,
liver damage was restricted to a few small necrotic areas. These studies
further indicated that Kupffer cells play an important role in T-cell

activation-induced liver injury by contributing tumor necrosis factor.
36
After administration of clodronate liposomes in nonobese diabetic
(NOD) mice, it was shown that T cells lost their ability to differentiate into
beta cell–cytotoxic T cells in a macrophage-depleted environment,
resulting in the prevention of autoimmune diabetes. These T cells regained
their beta cell–cytotoxic potential when they were returned to a macro-
phage-containing environment.
37
In these studies, the administration of
IL-12 seemed to reverse the prevention of diabetes, which was conferred
by macrophage depletion, in these NOD mice.
Gene Therapy
Replication-deficient recombinant adenovirus vectors are efficient at
transferring genes to target cells. However, both the innate immune system
and the acquired immune system may reduce the efficacy of this approach
for gene transfer. By their activity as scavengers of foreign particulate ma-
terial, macrophages may remove most of the injected gene carriers before
they can reach their targets.
35
P. G. Popovich, Z. Guan, P. Wei, I. Huitinga, N. van Rooijen, and B. T. Stokes, Exp.
Neurol. 158, 351 (1999).
36
J. Schumann, D. Wolf, A. Pahl, K. Brune, T. Papadopoulos, N. van Rooijen, and G. Tiegs,
Am. J. Pathol. 157, 1671 (2000).
37
H. S. Jun, C. S. Yoon, L. Zbytunik, N. van Rooijen, and J. W. Yoon, J. Exp. Med. 189, 347
(1999).
10 liposomes in immunology [1]
It has been shown that depletion of Kupffer cells by liposome-encapsu-

lated clodronate before intravenous administration of an adenovirus vector
led to a higher input of recombinant adenoviral deoxyribonucleic acid
(DNA) to the liver, an absolute increase in transgene expression, and a
delayed clearance of both the vector DNA and transgene expression.
One week after administration of the adenovirus vector, peak transgene
expression was found to be enhanced about 10-fold in the macrophage-
depleted animals. One month after administration, expression in the animals
treated with clodronate liposomes was still 38% of this peak value, whereas
control animals that got the adenovirus but not the liposomes showed no
detectable expression after 2 weeks.
38
Significantly higher and stable ex-
pression levels resulting from high-capacity adenovirus vectors that were
preceded by administration of clodronate liposomes have since been
reported in various models of gene therapy.
39,40
Also in the lung, alveolar
macrophages were shown to play an important role in the elimination of in-
tratracheally administered adenovirus vectors, and their suppressing effect
on adenovirus vector–mediated gene transfer could be eliminated by a
preceding intratracheal administration of clodronate liposomes.
41
The non-
linear dose response, following the application of adenovirus vectors for
gene therapy of Fabry disease in a mouse model, could be corrected by
the preceding administration of clodronate liposomes. As a consequence,
lower doses with strongly reduced toxicity are required.
42
These results
also suggest that minimizing the interaction between the recombinant

adenoviral vectors and the mononuclear phagocyte system may improve
the therapeutic window of this vector system.
42
Drug Targeting
Liposomes may be considered one of the most versatile and promising
drug-carrier devices (see the present and accompanying volumes). How-
ever, the high phagocytic capacity of tissue macrophages prevents the bulk
38
G. Wolff, S. Worgall, N. van Rooijen, W. R. Song, B. G. Harvey, and R. G. Crystal, J. Virol.
71, 624 (1997).
39
G. Schiedner, S. Hertel, M. Johnston, V. Dries, N. van Rooijen, and S. Kochanek, Molec.
Ther. 7, 35 (2003).
40
M. K. L. Chuah, G. Schiedner, L. Thorrez, B. Brown, M. Johnston, V. Gillijns, S. Hertel,
N. van Rooijen, D. Lillicrap, D. Collen, T. vanden Driessche, and S. Kochanek, Blood, 101,
1734 (2003).
41
S. Worgall, P. L. Leopold, G. Wolff, B. Ferris, N. van Rooijen, and R. G. Crystal, Human
Gene Ther. 8, 1675 (1997).
42
R. J. Ziegler, C. Li, M. Cherry, Y. Zhu, D. Hempel, N. van Rooijen, Y. A. Ioannou,
R. J. Desnick, M. A. Goldberg, N. S. Yew, and S. H. Cheng, Human Gene Ther. 13, 935
(2002).
[1] depletion of macrophages by liposomes 11
of all kinds of particulate carriers, including liposomes, to reach their
targets. Several modifications of the original liposome formulations, such
as the incorporation of amphipathic polyethylene glycol (PEG) conjugates
in the liposomal bilayers, have been proposed to reduce the recognition of
liposomes by macrophages. Nevertheless, a large percentage of these so-

called long-circulating liposomes will still be ingested by macrophages, as
has been shown in both the spleen
43
and lymph nodes.
44
Depletion of liver
and splenic macrophages by clodronate liposomes significantly prolonged
the circulation time of subsequently administered liposomes, even when
the latter were long-circulating liposomes. The efficacy of drug targeting
through the use of particulate carriers may thus be improved by the transi-
ent suppression of the phagocytic activity of macrophages by clodronate
liposomes.
When drug targeting is considered, repeated injections of the drug car-
riers will often be necessary to achieve the required effects. It has been
shown that long-circulating PEG-liposomes are cleared rapidly from the
circulation when injected repeatedly in the same animal. However, when
liver and splenic macrophages were previously depleted by clodronate lipo-
somes, such an enhanced clearance of repeatedly injected liposomes was
not observed,
45
emphasizing that suppression of phagocytic activity by clo-
dronate liposomes may contribute to the success of drug-carrier–mediated
therapy.
Materials and Methods
Preparation of Multilamellar Clodronate-Liposomes
1. Equipment and reagents
. Chloroform, analytical grade (Riedel-de Hae
¨
n, Seelze, Germany)
. Argon gas (or other inert gas, e.g., nitrogen gas)

. Sterile phosphate-buffered saline (PBS) (Braun Melsungen AG
Melsungen, Germany) containing 8.2 g NaCl, 1.9 g Na
2
HPO
4
Á2H
2
O,
0.3 g NaH
2
PO
4
Á2H
2
O at pH 7.4 per liter.
. Stock solution of phosphatidylcholine (egg lecitin): 100 mg/ml
phosphatidylcholine (Lipoid) in chloroform.
46
The solution is filtered
43
D. C. Litzinger, A. M. J. Buiting, N. van Rooijen, and L. Huang, Bioch. Bioph. 1190, 99
(1994).
44
C. Oussoren, M. Velinova, G. Scherphof, J. J. van der Want, N. van Rooijen, and G. Storm,
Bioch. Bioph. Acta, 1370, 259 (1998).
45
P. Laverman, M. G. Carstens, O. C. Boerman, E. Th. M. Dams, W. J. G. Oyen, N. van
Rooijen, F. H. M. Corstens, and G. Storm, J. Pharm. Exp. Ther. 298, 607 (2001).
12 liposomes in immunology [1]
with a syringe-driven filter unit with 0.2-m pores (Millex GN,

Millipore, Bradford, MA) on a glass/Teflon syringe.
. Stock solution of cholesterol: 10 mg/ml cholesterol (Sigma) in
chloroform.
47
The solution is filtered with a syringe-driven filter unit
with 0.2-m pores (Millipore, Millex GN) on a glass/Teflon syringe.
. 0.7 M clodronate solution: 50 g clodronate (Roche Diagnostics
GmbH Mannhein, Germany) is dissolved in 150 ml Milli Q (or
similar purified water). The pH is adjusted to 7.1 with 5 N NaOH.
The final volume is brought to 200 ml with Milli Q. This solution is
filtered with 0.2-m pore bottle-top filter (Millipore, Steritop).
. Waterbath sonicator (Sonicor SC-200-22, 55 kHz; Sonicor Instr.
Corp., Copiague, NY).
. High-speed centrifuge (Sorvall, RC 5B plus).
. Rotary evaporator (Bu
¨
chi, Rotavapor).
. Sterile pipets (Cellstar, Greiner Bio-One, Frickenhausen, Germany).
. Polycarbonate centrifuge tubes (Sorvall).
. Bottle-top filter 0.2-m pores (Millipore, Steritop).
. Autoclaved 3.0-m pore polycarbonate membrane filter (Millipore,
Isopore TSTP 2500) in filter holder (Millipore, Swinnex SX 2500).
2. Preparation of liposomes
. Forty-three milliliters of the phosphatidylcholine stock solution are
added to 40 ml cholesterol stock solution in a 2-liter round-bottom
flask.
48
. The chloroform is removed by low-vacuum (120 mbar) rotary
evaporation (150 rpm) at 40


. At the end, a thin phospholipid film
will form against the inside of the flask. The condensed chloroform is
removed, and the flask is aerated three times.
. The flask is vented by putting a pipet (without cotton-wool) at the
end of the argon gas tube. The tip of the pipet can be used to vent
deep into the flask to ensure ventilating the whole film and thus
removing all remaining chloroform.
. The phospholipid film is dispersed in 200 ml PBS (for empty
liposomes) or 0.7 M clodronate solution (for clodronate liposomes)
46
This stock can be made in advance and stored at À20

under argon gas. Argon gas is used to
prevent oxidation of phosphatidylcholine.
47
This stock can be made in advance and stored at À20

.
48
Instructions are given for preparation of 200 ml liposome suspension as we usually do.
However, smaller volumes can be made by reducing phosphatidylcholine, cholesterol, and
dispersing liquid. Attention should be paid to choose the most suitable filter, because the
amount of liquid loss depends on the diameter of the filter unit.
[1] depletion of macrophages by liposomes 13
by gentle rotation (max., 100 rpm) at room temperature (RT) for 20–
30 min (PBS) or 10–15 min (0.7 M clodronate solution).
49
Develop-
ment of foam should be avoided.
. The milky white suspension is kept at RT for 1.5–2h.

. The solution is shaked gently and sonicated in a waterbath for 3 min.
. The suspension is kept at RT for 2 h (or overnight at 4

) to allow
swelling of the liposomes.
50
. Before using the clodronate liposomes:
— The nonencapsulated clodronate is removed by centrifugation
of liposomes at 22,000g and 10

for 60 min. The clodronate
liposomes will form a white band at the top of the suspension,
whereas the suspension itself will be nearly clear.
51
— The clodronate solution under the white band of liposomes is
carefully removed using a sterile pipet (about 1% will be
encapsulated). The liposomes are resuspended in approximately
450 ml sterile PBS.
— The nonencapsulated clodronate is recycled for re-use. The
clodronate solution is centrifuged at 22,000g and 10

for
120 min. The remaining liposomes are discarded. This solution
is filtered using a 0.2-m bottle-top filter. This recycling
procedure should not be repeated for more than five times.
. The liposomes should be washed four to five times using centrifuga-
tion at 22,000g and 10

for 25 min. The upper solution should
be removed each time and the pellet resuspended in approximately

450-ml sterile PBS using a sterile pipet.
. The final liposome pellet is resuspended in sterile PBS and adjusted
to a final volume of 200 ml. The suspension is shaken (gently) before
administration to animals or before dispensing to achieve a
homogeneous distribution of the liposomes in suspension.
52
49
Clodronate liposomes can be stored in the original clodronate solution at 4

under argon
gas to prevent denaturation of phospholipid vesicles. This is particulary important in the
case of clodronate liposomes, because they float on the aqueous phase after preparation.
PBS liposomes form a pellet on the bottom of the tubes.
50
To limit the maximum diameter of the liposomes for intravenous injection, the suspension
can be filtered using membrane filters with 3.0-m pores.
51
There is no problem when the suspension is not completely clear, because the remaining
liposomes will be very small. The relatively large clodronate liposomes are efficacious with
respect to depletion of macrophages.
52
Sterility can be tested by distributing 50-m liposomes on a blood-agar plate.
14 liposomes in immunology [1]
Spectrophotometric Determination of the Amount of
Liposome-Encapsulated Clodronate
1. Equipment and reagents
. 3*1 ml of clodronate–liposome suspension to be tested (i.e., in
triplicate).
. Milli Q or similar purified water.
. Chloroform, analytical grade (Riedel-de Hae

¨
n).
. Sterile PBS (Braun Melsungen AG) containing 8.2 g NaCl, 1.9 g
Na
2
HPO
4
Á2H
2
O, 0.3 g NaH
2
PO
4
Á2H
2
O at pH 7.4 per liter.
. Standard clodronate solution (10.0 mg/ml): to prepare this, 500 mg
clodronate (Roche Diagnostics GmbH) is dissolved in 30-ml Milli Q.
The pH is adjusted to 7.1 with 5 N NaOH. The solution is brought to
a final volume of 50.0 ml with Milli Q.
. Phenol 90% (Riedel-de-Hae
¨
n, 16018).
. 4mM CuSO
4
solution: to prepare this, 0.64 g/liter CuSO
4
(Merck) is
dissolved in Milli Q.
. HNO

3
solution: to prepare this, 65% HNO
3
(Merck, Darmstadt,
Germany) is diluted 100 times in Milli Q.
. 16-ml glass tubes, caps with Teflon inlay (Kimble, Vineland, NY).
. 10-ml polystyrene tubes (Greiner).
. Spectrophotometer (UV-160A, Shimadzu, Kyoto, Japan).
. Pasteur pipets.
. Glass pipet 10 ml (piston pipet; Hirschmann).
. Pipets (P20, P200, and P1000, Gilson, Emeryville, CA).
2. Extraction of clodronate from liposomes
. 1 ml of the clodronate–liposome suspension (in triplicate), 1 ml of
standard clodronate solution, and 1 ml of the PBS solution is
dispensed in separate glass tubes.
53
. 8 ml of phenol/chloroform (1:2) is added to each tube.
. The tubes are vortexed and shaken extensively.
. The tubes are held at RT for at least 15 min.
. The tubes are centrifuged (1125 g)at10

for 10 min.
. The aqueous (upper) phase is transferred to clean glass tubes using a
Pasteur pipet.
. 6-ml chloroform per tube is added: the solution is reextracted by
extensive vortexing.
53
Attention should be paid to the right controls. If liposomes are suspended in PBS, PBS
controls should be included. nb: Phosphate (depending on concentration) may disturb the
assay.

[1] depletion of macrophages by liposomes 15
. The tubes are held for at least 5 min at RT.
. The tubes are centrifuged (1125 g)at10

for 10 min.
. The aqueous phase is transferred to 10-ml plastic tubes with a Pasteur
pipet. These are the samples for determination of clodronate
concentration.
3. Determination of clodronate concentration
. A standard curve using 0, 10, 20, 40, 50, 70, and 80 l of the extracted
standard clodronate solution adjusted with saline to a total volume of
1 ml per tube is prepared.
. The samples are diluted until they are within the range of the
standard curve.
54
. 2.25-ml 4 mM CuSO
4
solution, 2.20-ml Milli Q, and 0.05-ml HNO
3
solution is added to each tube, containing 1-ml sample or standard.
. All tubes are vortexed vigorously
. The samples are read at 240 nm using spectrophotometer and quartz
cuvette.
55
[2] Trafficking of Liposomal Antigens to the Trans-Golgi
Complex in Macrophages
By Mangala Rao,Stephen W. Rothwell, and Carl R. Alving
Introduction
The use of liposomes as potential carriers of antigens for vaccines in
combination with a variety of adjuvants and mediators has been explored

extensively,
1–4
and the first liposomal vaccine (for hepatitis A) was been
licensed in Europe
5,6
The major justification and rationale for using
54
A suspension of clodronate liposomes prepared according to protocol 1 contains about 6 mg
clodronate per 1 ml suspension. Twenty microliters of extracted clodronate liposome
suspension (thus diluting the sample 50 times) has an average absorption of 0.5 using a 1-cm
quartz cuvette.
55
J. Mo
¨
nkko
¨
nen, M. Taskinen, S. O. K. Auriola, and A. Urtti, J. Drug Targeting 2, 299
(1994).
1
C. R. Alving, V. Koulchin, G. M. Glenn, and M. Rao, Immunol. Rev. 145, 5 (1995).
2
C. R. Alving, J. Immunol. Meth. 140, 1 (1991).
3
G. Gregoriadis, Immunol. Today 11, 89 (1990).
4
L. F. Fries, D. M. Gordon, R. L. Richards, J. E. Egan, M. R. Hollingdale, M. Gross,
C. Silverman, and C. R. Alving, Proc. Natl. Acad. Sci. U S A 89, 358 (1992).
5
L. Loutan, P. Bovier, B. Althaus, and R. Glu
¨

ck, Lancet 343, 322 (1994).
METHODS IN ENZYMOLOGY, VOL. 373 0076-6879/03 $35.00
16 liposomes in immunology [2]
. The tubes are held for at least 5 min at RT.
. The tubes are centrifuged (1125 g)at10

for 10 min.
. The aqueous phase is transferred to 10-ml plastic tubes with a Pasteur
pipet. These are the samples for determination of clodronate
concentration.
3. Determination of clodronate concentration
. A standard curve using 0, 10, 20, 40, 50, 70, and 80 l of the extracted
standard clodronate solution adjusted with saline to a total volume of
1 ml per tube is prepared.
. Thesamplesarediluteduntiltheyarewithintherangeofthe
standardcurve.
54
. 2.25-ml 4 mM CuSO
4
solution, 2.20-ml Milli Q, and 0.05-ml HNO
3
solution is added to each tube, containing 1-ml sample or standard.
. All tubes are vortexed vigorously
. The samples are read at 240 nm using spectrophotometer and quartz
cuvette.
55
[2] Trafficking of Liposomal Antigens to the Trans-Golgi
Complex in Macrophages
By Mangala Rao,Stephen W. Rothwell, and Carl R. Alving
Introduction

The use of liposomes as potential carriers of antigens for vaccines in
combination with a variety of adjuvants and mediators has been explored
extensively,
1–4
and the first liposomal vaccine (for hepatitis A) was been
licensed in Europe
5,6
The major justification and rationale for using
54
A suspension of clodronate liposomes prepared according to protocol 1 contains about 6 mg
clodronate per 1 ml suspension. Twenty microliters of extracted clodronate liposome
suspension (thus diluting the sample 50 times) has an average absorption of 0.5 using a 1-cm
quartz cuvette.
55
J. Mo
¨
nkko
¨
nen, M. Taskinen, S. O. K. Auriola, and A. Urtti, J. Drug Targeting 2, 299
(1994).
1
C. R. Alving, V. Koulchin, G. M. Glenn, and M. Rao, Immunol. Rev. 145, 5 (1995).
2
C. R. Alving, J. Immunol. Meth. 140, 1 (1991).
3
G. Gregoriadis, Immunol. Today 11, 89 (1990).
4
L. F. Fries, D. M. Gordon, R. L. Richards, J. E. Egan, M. R. Hollingdale, M. Gross,
C. Silverman, and C. R. Alving, Proc. Natl. Acad. Sci. U S A 89, 358 (1992).
5

L. Loutan, P. Bovier, B. Althaus, and R. Glu
¨
ck, Lancet 343, 322 (1994).
METHODS IN ENZYMOLOGY, VOL. 373 0076-6879/03 $35.00
16 liposomes in immunology [2]
liposomes as vehicles for vaccines has been the rapid uptake of liposomes
by macrophages.
7–9
In this chapter, we describe methods for examining the
intracellular fate of liposomes and liposomal antigens in macrophages.
Protein antigens are processed and presented either by the major histo-
compatibility complex (MHC) class I or class II pathways.
10,11
MHC class I
molecules are expressed on the surface of all nucleated cells. In contrast,
MHC class II molecules are expressed only on the surface of antigen pre-
senting cells (APCs), such as macrophages, B cells, and dendritic cells.
MHC class I and class II molecules are highly polymorphic membrane
proteins that bind and transport peptide fragments of proteins to the sur-
face of APCs. The MHC–peptide complex then interacts with either
CD8
+
or CD4
+
T lymphocytes to generate a specific immune response.
10,12
Endogenous antigens are presented by way of the MHC class I path-
way, whereas exogenous antigens are presented by way of the MHC class
II pathway. Therefore, most soluble antigens are relatively ineffective for
priming MHC class I–restricted cytotoxic T lymphocyte responses because

of the inability of the antigen to gain access to the cytoplasmic compart-
ment. Several different methods have been used to channel antigens into
the class I pathway.
1,2, 13–23
Among these methods, liposomes have proven
6
R. Glu
¨
ck, Vaccine 17, 1782 (1999).
7
D. Su and N. Van Rooijen, Immunology 66, 466 (1989).
8
J. N. Verma, M. Rao, S. Amselem, U. Krzych, C. R. Alving, S. J. Green, and N. M. Wassef,
Infect. Immun. 60, 2438 (1992).
9
J. N. Verma, N. M. Wassef, R. A. Wirtz, C. T. Atkinson, M. Aikawa, L. D. Loomis, and
C. R. Alving, Biochim. Biophys. Acta 1066, 229 (1991).
10
R. N. Germain and D. H. Margoulies, Ann. Rev. Immunol. 11, 403 (1993).
11
T. J. Braciale, L. A. Morrison, M. T. Sweetser, J. Sambrook, M. J. Gething, and
V. L. Braciale, Immunol. Rev. 98, 95 (1987).
12
A. Townsend and H. Bodmer, Ann. Rev. Immunol. 7, 601 (1989).
13
M. W. Moore, F. R. Carbone, and M. J. Bevan, Cell 54, 777 (1988).
14
K. Deres, H. Schild, K. H. Weismuller, G. Jung, and H. G. Rammensee, Nature 342, 561
(1989).
15

H. Schild, M. Norda, K. Deres, K. Falk, O. Rotzschke, K. H. Weismuller, G. Jung, and
H. G. Rammensee, J. Exp. Med. 174, 1665 (1991).
16
C. V. Harding and R. Song, J. Immunol. 153, 4925 (1994).
17
M. Kovacsovics-Bankowski and K. L. Rock, Science 267, 243 (1995).
18
Y. Men, H. Tamber, R. Audran, B. Gander, and G. Corradin, Vaccine 15, 1405 (1997).
19
L. M. Lopes and B. M. Chain, Eur. J. Immunol. 22, 287 (1992).
20
R. Reddy, F. Zhou, S. Nair, L. Huang, and B. T. Rouse, J. Immunol. 148, 1585 (1992).
21
K. White, U. Krzych, T. D. Gordon, M. R. Porter, R. L. Richards, C. R. Alving, C. D. Deal,
M. Hollingdale, C. Silverman, D. R. Sylvester, W. R. Ballou, and M. Gross, Vaccine 11,
1341 (1993).
22
W. I. White, D. R. Cassatt, J. Madsen, S. J. Burke, R. M. Woods, N. M. Wassef, C. R. Alving,
and S. Koenig, Vaccine 13, 1111 (1995).
23
C. R. Alving and N. M. Wassef, AIDS Res. Hum. Retrovir. 10, S91 (1994).
[2] trafficking of liposomal antigens 17
to be an efficient delivery system for entry of exogenous protein antigens
into the MHC class I pathway because of their particulate nature.
24
A lipo-
some formulation developed in our laboratory that contains dimyristoyl
phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol, and
an encapsulated protein antigen has been used in human clinical trials.
4

This formulation of liposomes has also been shown to be an effective
vehicle for delivery of proteins or peptides to APCs for presentation by
way of the MHC class I pathway in mice.
25,26
By the use of fluorophore-labeled proteins encapsulated in liposomes,
we have addressed the question of how liposomal antigens enter the
MHC class I pathway in bone marrow–derived macrophages. After phago-
cytosis of the liposomes, the liposomal lipids and the liposomal proteins
seem to follow the same intracellular route, and they are processed as a
protein-lipid unit.
27
The fluorescent liposomal protein and liposomal lipids
enter the cytoplasm where they are processed by the proteasome com-
plex.
25
The processed liposomal protein is then transported into the endo-
plasmic reticulum and the Golgi complex by way of the transporter
associated with antigen processing (TAP).
28
In these compartments, the
peptides bind to the MHC class I molecules. Once bound, the antigenic
peptides are transported to the cell surface to interact with receptors on
T cells.
29,30
The procedures that we use to study the intracellular trafficking
of liposome-encapsulated proteins are outlined in the following.
Experimental Design
We have developed an in vitro antigen presentation system consisting of
bone marrow–derived macrophages as the APCs. Our system is well suited
for studying intracellular trafficking, because we begin with precursor cells

that can be differentiated into either dendritic cells or macrophages.
Although B cells can also be used to study intracellular trafficking of anti-
gens, we have used bone marrow–derived macrophages because of the ease
of preparation, their inherent phagocytic properties, their ability to adhere to
24
M. Rao and C. R. Alving, Adv. Drug Deliv. Rev. 41, 171 (2000).
25
S. W. Rothwell, N. M. Wassef, C. R. Alving, and M. Rao, Immunol. Lett. 74, 141 (2000).
26
R. L. Richards, M. Rao, N. M. Wassef, G. M. Glenn, S. W. Rothwell, and C. R. Alving,
Infect. Immun. 66, 2859 (1998).
27
M. Rao, S. W. Rothwell, N. M. Wassef, A. B. Koolwal, and C. R. Alving, Exp. Cell Res. 246,
203 (1999).
28
M. Rao, S. W. Rothwell, N. M. Wassef, R. E. Pagano, and C. R. Alving, Immunol. Lett. 59,
99 (1997).
29
C. Bonnerot, V. Briken, and S. Amigorena, Immunol. Lett. 57, 1 (1997).
30
S. Joyce, J. Mol. Biol. 266, 993 (1997).
18 liposomes in immunology [2]